Intracellular signaling pathways depend upon appropriate and unique subcellular locations of their constituent proteins. Frequently, key intracellular signaling proteins move from one subcellular location to another in response to extracellular stimuli. Incorrectly localized proteins can prevent completion of a particular signaling pathway or can cause unregulated signaling, contributing to disease states such as cancer and hypertrophy of the heart. Understanding how cellular proteins come together at specific times and subcellular sites will lead to insight into ways to block inappropriate signaling in disease. Mechanisms responsible for reversibly targeting peripheral membrane proteins to different cellular membranes are poorly understood. Moreover, it is not clear how different subcellular localizations influence a signaling protein's function. This applicaton will focus on several key questions regarding the mechanisms and function of intracellular trafficking of heterotrimeric (a [unreadable] ?) G proteins. G proteins act as molecular switches to relay information from cell surface receptors to appropriate effector proteins. To transmit a signal from a G protein-coupled receptor (GPCR), G proteins must be localized to the cytoplasmic face of the plasma membrane (PM). However, it is becoming increasing clear that G proteins are not statically localized at the PM, but instead follow distinct trafficking pathways to reach the PM and internalize and recycle to the PM in response to GPCR activation. Additionally, new findings suggest that G proteins have important, yet poorly defined, signaling functions at subcellular locations other than the PM. To address these issues, the major objectives of this proposal are 1) Define pathways of endomembrane to PM trafficking of Ga and G[unreadable]?; 2) Define trafficking pathways and recycling mechanisms of GPCR-activated Ga and G[unreadable]? Define subcellular location of palmitoylation of Ga and define novel signaling functions of Ga and G[unreadable]? at endomembranes. This research will utilize cultured mammalian cells as model systems and will employ a number of techniques, include immunofluorescence microscopy, fluorescence microscopy of live cells, subcellular fractionation, and numerous biochemical assays to define structure-function relationships in terms of mechanisms of reversible G protein trafficking.